The long-term objective of this project is to elucidate mechanisms supporting acute modulation of the norepinephrine (NE) transporter (NET) as established by coordinated trafficking and intrinsic activity modulation. NE signaling at PNS and CNS noradrenergic synapses regulates a variety of physiological functions including attention, motivation, vasoconstriction and heart rate. The NET dictates the magnitude and duration of NE signaling. For decades, the main focus on NET has been as a target for antidepressant and psychostimulant (e.g. amphetamine (AMPH), cocaine, and methylphenidate (Ritalin)) action. Our recent findings indicate that the stability of NET protein-protein associations, including interactions with syntaxin (SYN) 1A and protein phosphatase 2A (PP2A), dictate NET cell surface distribution and NET intrinsic activities, including transport rates, NET-gated currents and efflux potential. This renewal application unites two laboratories at the forefront of neurotransmitter transporter research, linking methods in biochemistry and molecular biology with real-time imaging and biophysical approaches, and includes studies on neuronal cell lines expressing NET and NET mutants as well as an evaluation of native noradrenergic neurons. We focus our efforts on three distinct, but coordinated, aspects of modulated NET behavior: the exocytosis of NET proteins, the endocytosis of NET proteins and the modulation of NET associated ion/substrate flux properties. Our specific aims are to: 1) Determine whether activity-dependent changes in NET surface density reflect increased insertion of cytoplasmic transporters or reduced transporter endocytosis (or both) and define the sites supporting SYN 1A/PP2Ac associations and their functional consequences with respect to NET localization and surface insertion; 2) Elucidate structural and signaling determinants of NET endocytic trafficking as triggered by GPCRs and AMPH; and 3) Determine relationships between NET/associated proteins/Ca2+ and AMPH-triggered NE efflux. Our overall goal then is to develop a more sophisticated understanding of the nature of acute NET regulation and to determine how the formation/dissolution of NET protein complexes promotes the coordination of NE clearance with NE release and clarifies intrinsic mechanisms by which psychostimulants impact NET mediated ion and NE flux.